In the past several years, there has been a tremendous increase of interest in using RNA interference (RNAi) for biomedical applications. RNAi is a posttranscriptional sequence specific process of gene silencing employing double-stranded RNAs (dsRNAs) and a set of specific proteins and enzymes. To briefly explain the mechanism, the RNaseIII-like enzyme, Dicer, processes a dsRNAs into shorter duplexes (21-23 bp). These duplexes, referred to as short interfering RNAs (siRNAs), are then loaded into a RNA-induced silencing complex (RISC) and one of the siRNA strands, called passenger or sense, is discarded. The other strand, called guide or antisense, is used by RISC to recognize the target mRNA for cleavage and translation prevention. RNAi has become a powerful technique for selective suppression of particular genes of interest in different species showing potential for use as cancer and HIV therapeutics. Synthetic siRNAs against particular genes of interest can be exogenously introduced into cells to activate RNAi. Moreover, introduction of synthetic asymmetric Dicer substrates slightly longer than siRNAs (25 bp) tremendously increases the potency of silencing. This can be explained by the involvement of Dicer in the process of loading the RISC complex with siRNAs. Despite the potential for siRNA, there is a need for novel approaches that overcome several challenges associated with the clinical delivery of RNAi.
As described herein, the present invention splits the functionality of Dicer substrates siRNA duplexes into two R/DNA hybrids, which upon simultaneous presence inside the same diseased cell will recognize each other through toehold interaction and re-associate releasing active siRNAs. This novel approach will overcome several challenges associated with the clinical delivery of RNAi, such as intravascular degradation (will be reduced for R/DNA hybrids), tissue specificity (DNA chemistry is more parsimonious than RNA and amenable to chemical modifications with different features for targeting or delivery), pharmacodynamics (fluorescent tags can be activated upon R/DNA hybrid re-association assisting in Förster resonance energy transfer (FRET) imaging of delivery and response). Moreover, all these additional functionalities can be introduced through chemical modifications of the DNA strands in the R/DNA hybrids thus, not interfering with the processivity of the released siRNAs. Additionally, the number of these functionalities can be at least as large as twice the number of DNA strands entering into the composition of the duplex hybrids or more complex hybrid nanostructures.